C-terminal proteolysis of the avian 1,25-dihydroxyvitamin D₃ receptor

Exposure of the 60 kDa chick intestinal 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) receptor to carboxypeptidase A resulted in a time dependent decrease in receptor hormone-binding; after 2 h, there was no detectable macro-molecular-bound 1,25(OH)₂[³H]D₃. Upon DNA-cellulose chromatography of this preparation, a 56 kDa protein adsorbed to the column and eluted as a function of para-chloromercuribenzene sulfonate (a sulfhydryl blocking reagent). The 56 kDa fragment was detected by anti-receptor monoclonal antibodies via immunoblot technology. The 1,25(OH)₂[³H]D₃ eluted in the fall through fractions of the column. Thus, cleavage of up to 40 amino acids from the carboxy-terminus of the 1,25(OH)₂D₃ receptor results in a protein which no longer binds to hormone, but retains its capacity to interact with DNA-cellulose and monoclonal antibody. These results represent novel biochemical evidence that allows us to orient the 1,25(OH)₂D₃ binding domain near the C-terminus of the receptor.